The long-term goal of the proposed studies is to understand the mechanisms that determine longevity. Animal species differ enormously in their aging rates. Therefore, a comparative approach is a powerful tool to obtain new insights into the mechanisms of aging. Accumulation of somatic mutations and genomic rearrangements is believed to be a contributing cause of aging. Data collected from mouse studies suggest that DNA double-strand break (DSB) repair is an essential longevity assurance mechanism. Considering recent advances in understanding DNA DSB repair [and DSB response mechanisms], we believe that it is timely to undertake an interspecies comparative study of DNA DSB repair [and DSB response]. In this application we propose to test the hypothesis that genome maintenance mechanisms co evolve with lifespan, using a panel of rodent species. Rodents are an ideal species for this study as it includes phylogenetically related species with diverse lifespans, in which slow aging has evolved independently several times. Our laboratory has assembled a collection of primary cells and tissues from 17 rodent species, and performed preliminary analysis of telomerase activity, cell growth characteristics, and genome stability in these cells. Our preliminary data suggest that long-lived species have higher genome stability and more efficient DSB repair. In this application, we will employ the collection of primary rodent cells and tissues to: (1) Test the hypothesis that genome stability coevolves with long lifespan. We will examine aneuploidy rate by flow cytometry and analyze specific chromosomal aberrations by karyotyping. (2) Test the hypothesis that the efficiency and fidelity of DSB repair co evolve with lifespan. We have recently developed sensitive fluorescent assays for analysis of the efficiency and fidelity of the two pathways of DSB repair, homologous recombination (HR) and nonhomologous end joining (NHEJ). These assays will enable quantitative analysis of DSB repair in multiple species. (3) Test the hypothesis that specific DSB responses such as apoptosis and senescence co evolve with lifespan. We will measure cell survival, induction of DSB repair foci, apoptosis, necrosis, and senescence following y-irradiation. We will also analyze the rate DSB repair by comet assays. The data will be corrected for phylogenetic nonindependence, and controlled for confounding variables such as body size. Our laboratory is uniquely equipped to carry out the proposed research. We assembled a carefully constructed collection of phylogenetically related species with diverse lifespans, and developed novel quantitative assays for the analysis of HR and NHEJ, and sensitive assays for analysis of apoptosis and necrosis. The proposed study will determine whether such genome maintenance mechanisms as induction of apoptosis, senescence, or the efficiency and fidelity of DSB repair co evolve with lifespan. It will also identify molecular mechanisms responsible for the differences in genome maintenance between short- and long-lived rodents. PUBLIC HEALTH RELEVANCE: The mechanisms responsible for the vast differences in lifespan between animal species are unknown. The proposed project seeks to examine whether genome maintenance mechanisms contribute to interspecies differences in longevity using a collection of short- and long-lived rodent species. The information obtained in this study will help to develop strategies to prevent cancer and extend lifespan in humans.